The nonstructural p17 protein of a fusogenic bat-borne reovirus regulates viral replication in virus species- and host-specific manners

Nelson Bay orthoreovirus (NBV), a member of the family Reoviridae, genus Orthoreovirus, is a bat-borne virus that causes respiratory diseases in humans. NBV encodes two unique nonstructural proteins, fusion-associated small transmembrane (FAST) protein and p17 protein, in the S1 gene segment. FAST induces cell–cell fusion between infected cells and neighboring cells and the fusogenic activity is required for efficient viral replication. However, the function of p17 in the virus cycle is not fully understood. Here, various p17 mutant viruses including p17-deficient viruses were generated by a reverse genetics system for NBV. The results demonstrated that p17 is not essential for viral replication and does not play an important role in viral pathogenesis. On the other hand, NBV p17 regulated viral replication in a bat cell line but not in other human and animal cell lines. Nuclear localization of p17 is associated with the regulation of NBV replication in bat cells. We also found that p17 dramatically enhances the cell–cell fusion activity of NBV FAST protein for efficient replication in bat cells. Furthermore, we found that a protein homologue of NBV p17 from another bat-borne orthoreovirus, but not those of avian orthoreovirus or baboon orthoreovirus, also supported efficient viral replication in bat cells using a p17-deficient virus-based complementation approach. These results provide critical insights into the functioning of the unique replication machinery of bat-borne viruses in their natural hosts.


Introduction
Emerging viruses that spillover from their natural reservoirs are a serious threat to public health [1,2]. These viruses can cause lethal disease in humans without affecting the health of their natural host. For example, avian influenza virus H5N1 can cause severe respiratory disease with a high mortality rate in humans, but not in duck species [3], and hantavirus can cause hemorrhagic fever in humans, but is asymptomatic when it infects rodents [4,5]. Bats are thought to be the natural reservoirs for a number of deadly zoonotic viruses, including severe acute respiratory syndrome (SARS) coronaviruses, Ebola virus, and Nipah virus [6][7][8], and are regarded as the most likely natural host for the COVID-19 infectious agent, SARS-CoV-2 [9][10][11]. While no mortality or morbidity has been observed in bats experimentally infected with Hendra virus, Nipah virus, Marburg virus, Ebola virus, or SARS-like coronaviruses [12][13][14][15], the precise mechanisms that allow these viruses to circumvent pathogenesis in bats remain to be elucidated. These observations suggest that bat-borne viruses build symbiotic relationships in their reservoir hosts to regulate viral replication [8]. Such relationships between virus and host are thought to be established by complex mechanisms regulated by antiviral immune defenses and viral factors. Constitutive expression of interferon (IFN)-α has been observed in unstimulated bat tissue and cultured cells, and it is possible that constitutively expressed IFN and IFN-stimulated genes control viral replication in bat species [16]. It has similarly been reported that stimulator of IFN genes (STING), which is an important innate immune response protein involved in DNA sensing, and NLR family pyrin domain containing (NLRP) 3, which activates inflammatory mediators, are dampened in bats [17,18], contributing to a reduced inflammatory response and disease tolerance. However, although much is known about antiviral immune defenses in bats, the viral factors responsible for the control of replication of bat-borne viruses in their natural reservoirs remain largely unknown.
Nelson Bay orthoreovirus (NBV), a member of the genus Orthoreovirus belonging to the family Reoviridae, was first isolated in a fruit bat in Australia in 1968 [19]. Several NBV strains have since been isolated from different bat species [20][21][22][23] and, therefore, bats have been suggested as the natural reservoir for NBVs. Recently, NBVs have been isolated from patients with acute respiratory infection in several Asian countries [24][25][26]. In Japan, a pathogenic NBV Miyazaki-Bali/2007 (MB) strain was isolated from a Japanese patient who traveled to Bali, Indonesia [27]. These findings suggest that NBV has evolved to cross the species barrier from bats to humans. Members of the genus Orthoreovirus are divided into fusogenic and nonfusogenic subgroups, based on their ability to induce cell-cell fusion [28]. The orthoreovirus genome consists of ten segments of double-stranded RNA (dsRNA) (L1-L3, M1-M3, and S1-S4 gene segments) and some fusogenic reovirus genomes, including avian orthoreovirus (ARV), baboon orthoreovirus (BRV), Broome orthoreovirus (BroV), and NBV, encode two unique nonstructural proteins, the fusion-associated small transmembrane (FAST) protein and p17, on the S1 or S4 gene segment [29][30][31][32]. FAST proteins are small fusogenic proteins of approximately [10][11][12][13][14][15][16][17][18][19][20][21][22] kDa that can induce cell-cell fusion between infected-cells and neighboring cells [32,33]. FAST proteins are composed of three functional domains associated with fusion activity: an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain [34,35]. Unlike the structural proteins of enveloped viruses, nonstructural FAST proteins are dispensable for viral entry [36,37]. While the biological function of FAST proteins is poorly understood, it has been demonstrated that FAST is required for efficient viral propagation in vitro and plays a crucial role in pathogenesis in vivo [37].
Nonstructural NBV p17, ARV p17, BRV p16, and BroV p16 proteins are encoded by the second open reading frame of the fusogenic reovirus S1 or S4 gene segment [30,31,38,39]. Previous studies have revealed that ARV p17, also known as CRM1-independent nucleocytoplasmic shuttling protein, possesses a nuclear localization signal with critical C-terminal basic amino acids at residues K122 and R123 conserved among ARV and NBV strains [40]. In addition, it has been shown that ARV p17 activates the p53 signaling pathway and downregulates the PI3K/AKT/mTOR and ERK pathways [41]. The modulation of these fundamental signaling pathways results in the disruption of cellular translation, cell cycle arrest, and the formation of autophagosomes, contributing to efficient viral replication [41][42][43][44]. However, the biological functions of NBV p17 in the viral life cycle, and its involvement in viral pathogenesis, remain unclear.
In this study, we examined the roles of NBV p17 in viral replication in cell lines and viral pathogenesis in a mouse model by using p17-mutant and -deficient viruses. We found that replication of p17-deficient virus was severely impaired in cell lines derived from the fruit bat Rousettus leschenaultii, a potential natural host for NBV. Expression of NBV p17 and BroV p16, but not other p17 homologues, rescued the cell-cell fusion activity of NBV FAST, leading to efficient replication in bat cells, suggesting that p17 regulates viral replication in virus species-and host-specific manners.

NBV p17 is not required for viral replication in vitro
To understand the function of NBV p17 during the viral life cycle, we generated a p17-deficient mutant virus, using a reverse genetics system, in the pathogenic NBV strain MB [27,45]. A viable p17-null virus (rsMB/p17-null) was rescued from cells transfected with plasmids containing cDNA from nine NBV MB gene segments and S1 cDNA featuring a disrupted p17 translational start codon ( 277 ATG 279 !ACG) and a stop codon ( 301 TTA 303 !TAA) (Fig 1A and S1 Table). The genomic dsRNA pattern of rsMB/p17-null showed the same pattern as that of wild-type recombinant strain MB (rsMB) by electrophoresis (Fig 1B). To examine whether p17 is expressed in cells infected with rsMB/p17-null, we confirmed viral protein expression by immunoblotting protein extracts derived from infected cells. We found that while p17 expression was not detectable in cells infected with p17-null virus, the expression of NBV proteins, μNS (a component of the viral inclusion body where virus replication occurs) and σNS (a component of viral inclusion body) encoded by M3 and S3 segments, respectively, remained unchanged between p17-null virus-and wild-type virus-infected cells (Fig 1C). Expression of σC (cell-attachment protein) located downstream of the p17 ORF was increased in cells infected with the p17-null virus. The result is consistent with a previous report [46]. To

Fig 1. Evaluation of viral replication following wild-type and p17-null virus infection. (A)
Schematic presentation of the S1 gene segment of rsMB and rsMB/p17-null. Three ORFs, FAST (27-314 nt), p17 (277-705 nt), and σC (572-1567 nt), are encoded on the NBV S1 gene segment. In the p17-null virus, the p17 start codon is disrupted with a 277 ATG 279 !ACG substitution, and a stop codon introduced by a 301 TTA 303 !TAA substitution. (B) The electropherotype of the genomic RNA of rsMB and rsMB/p17-null. RNA extracted from virions was electrophoresed on a 10% polyacrylamide gel. (C) Electrophoretic analysis of viral proteins of rsMB and rsMB/p17-null. Vero cells were infected with rsMB or rsMB/p17-null at an MOI of 5 PFU/cell and lysed at 18.5 h post-infection. Cell lysates were subjected to immunoblotting using antibodies raised against NBV p17, NBV σC, NBV μNS, NBV σNS, or β-actin. (D) Growth kinetics of rsMB and rsMB/p17-null in Vero cells. The cells were infected with rsMB or rsMB/p17-null at an MOI of 3 PFU/cell (n = 3). Viral titers were determined by plaque assay using L929 cells. (E) Viral titers of rsMB and rsMB/p17-null in human and animal cell lines. Cells were infected with rsMB or rsMB/p17-null at an MOI of 0.1 PFU/ cell and collected at 72 h post-infection (n = 3). Viral titers were determined by plaque assay using L929 cells. Results are expressed as the mean of three samples. Error bars indicate standard deviations. ns, not significant (Welch's t-test, p > 0.01). https://doi.org/10.1371/journal.ppat.1010553.g001

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Regulation of viral replication by a viral factor in bat cells examine whether p17 plays a critical role in viral replication in cultured cells, wild-type and p17-null viruses were infected into various cell lines, and viral titers were determined by plaque assay. The growth kinetics of rsMB and rsMB/p17-null were similar in Vero cells ( Fig 1D) and in cell lines from humans and other animal species, such as, mouse, hamster, monkey, cow, dog, pig, and quail ( Fig 1E). These data indicate that p17 is not essential for viral replication and does not affect viral propagation in these human or animal cell lines.

NBV p17-deficient virus causes lethal infection in an NBV mouse model
In a previous study, we developed a mouse model for lethal NBV lung infection, which revealed that NBV proteins, σC and FAST, were virulence factors [37,45,47]. To understand the importance of p17 in viral pathogenesis, we intranasally inoculated 4-week-old C3H mice with either rsMB or rsMB/p17-null virus. No significant difference in body weight or survival rate was observed between mice infected with the rsMB virus and those infected with the rsMB/p17-null virus (Fig 2A and 2B), suggesting that p17 does not play an important role in viral pathogenesis in this model.

NBV p17 regulates viral replication in the bat cell line DemKT1
Recent epidemiological studies have reported that NBV infects several species of bat in their natural habitat, including Rousettus leschenaultii, suggesting bats as the presumptive natural reservoirs of NBV [20][21][22][23]48]. Thus, we hypothesized that p17 protein could have specific functions in this natural host. To understand the importance of p17 in bats, we infected a bat cell line derived from Rousettus leschenaultii, DemKT1 [49], with either rsMB or rsMB/ p17-null virus. Interestingly, the growth kinetics of rsMB/p17-null were significantly lower than those of wild-type rsMB (approximately 120-fold at 72 h post-infection) (Fig 3A). DemKT1 cells infected with rsMB/p17-null displayed improved cell viability compared with cells infected with rsMB (S1 Fig). The expression of several NBV proteins, including μNS, σA (inner capsid protein encoded by the S2 segment), and σC, was lower at 16 h post-infection in cells infected with the rsMB/p17-null than in those infected with wild-type rsMB ( Fig 3B).

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Regulation of viral replication by a viral factor in bat cells

Regulation of viral replication by a viral factor in bat cells
Together, these data indicated that p17 plays a critical role in the regulation of viral replication in the bat DemKT1 cell line. To investigate whether the function of p17 in viral replication observed in this bat cell line is conserved among other NBV strains, we generated monoreassortant viruses containing the S1 segment from two other NBV strains, Nelson Bay (NelB), a strain isolated from bats, and Melaka (Mel), a strain isolated from human, in an otherwise MB genetic background (rsMB/NelB-S1 and rsMB/Mel-S1, respectively). p17-deficient viruses were generated in these monoreassortant strains, as described above (S1 Table), resulting in the generation of rsMB/NelB-S1/p17-null and rsMB/Mel-S1/p17-null mutants. The replication kinetics of p17-null monoreassortants were similar to those of monoreassortant viruses expressing wild-type p17 in Vero cells ( Fig 3C); however, the replication kinetics were reduced 325-and 516-fold in DemKT1 cells upon infection with rsMB/Mel-S1 and rsMB/NelB-S1, respectively ( Fig 3D). These results suggest that the function for NBV p17 in the regulation of viral replication in bat cells is conserved among NBV strains.
The K117, R118, K131, and R132 amino acid residues of NBV p17 are important for nuclear localization and replication in DemKT1 cells p17 K131 and R132 are conserved in NBV strains, but not ARV strains ( Fig 5A). However, a second basic amino acid motif located adjacent to K131 and R132 is conserved among both NBV and ARV strains (K117 to R118 in NBV, and K122 to R123 in ARV) ( Fig 5A). It has previously been reported that ARV p17 K122A and R123A mutants impair nuclear localization [40], and we hypothesized that NBV p17 K117, R118, K131, and R132 could similarly be required for nuclear localization and the regulation of viral replication. To determine the importance of the two basic amino acid motifs in viral replication, we named the upstream K117 and R118 motif as uKR and downstream K131 and R132 motif as dKR, substituted each pair of amino acids with alanine residues in transient expression vectors, and examined the distribution of p17 in DemKT1 cells. While native p17 protein was found solely in the nucleus of transfected cells by immunoblotting ( Fig 5B) and immunofluorescence analysis (Fig 5C), alanine substitution of uKR and/or dKR led to an accumulation of p17 in the cytoplasm. These data suggest that both p17 basic amino acid motifs are required for nuclear localization. To investigate the relationship between nuclear localization of p17 and viral replication in

Regulation of viral replication by a viral factor in bat cells
DemKT1 cells, we generated p17 mutant viruses in which uKR and/or dKR were substituted to alanine and examined the growth of these viruses using the plaque assay in Vero and DemKT1 cells. The p17 uKR and dKR mutants replicated efficiently in Vero cells (Fig 5D), while viral titers were significantly lower in DemKT1 cells ( Fig 5E). These results suggest that nuclear localization of p17 is essential for efficient viral replication in DemKT1 cells.

The transient expression of p17 complements the replication defect of rsMB/p17-null virus in DemKT1 cells
p17 alanine substituted mutations in uKR and/or dKR motifs also introduced mutations in the N-terminus of the overlapping σC ORF (S1 Table). Thus, it is possible that the replication defect of p17 uKR and dKR mutants in DemKT1 cells was caused by modification of σC function. Therefore, we developed a p17 complementation system to examine whether p17 is directly responsible for efficient viral replication. DemKT1 cells were transfected with wildtype or mutant p17 expression plasmids and subsequently infected with rsMB/p17-null virus at an MOI of 1 or 0.1 PFU/cell at 10 h or 24 h post-transfection. Expression of wild-type p17 in trans rescued rsMB/p17-null viral growth and the expression of the viral proteins μNS, σC, σA, and σNS (Fig 6A and 6B). By contrast, all p17 constructs encoding uKR and dKR motif mutations failed to restore rsMB/p17-null virus replication (Fig 6A and 6B). We also observed that replication of the p17-null virus was restored by transient transfection with FLAG-tagged wild type p17 but not its mutants (K131A-FLAG, R132A-FLAG, uKR-AA-FLAG, dKR-AA-FLAG, and udKR-AA-FLAG) in DemKT1 cells (Fig 6C and 6D). These results confirm that nuclear localization of p17 plays a critical role in the regulation of NBV replication in DemKT1 cells.

Expression of NBV p17 is required for induction of fusion activity of FAST in bat cell lines
Both the wild-type rsMB and rsMB/p17-null viruses encode intact FAST protein in the S1 gene segment. FAST proteins are the only known fusogenic proteins found in non-enveloped viruses and can induce cell-cell fusion in infected cells [33,50]. Recently, we demonstrated that although FAST is not essential for viral replication, it can enhance replication and pathogenesis [37]. Interestingly, in the present study, we found that the rsMB/p17-null mutant virus induced fewer cell-cell fusion events than wild-type rsMB in DemKT1 cells, while cell-cell fusion was unaffected in Vero cells (Fig 7A). These data suggest that NBV p17 may regulate FAST function during viral replication in DemKT1 cells. Therefore, we next examined the fusion activity of FAST in bat cell lines transfected with p17. In Vero cells, expression of FAST induced the formation of large multi-nuclear cells, termed syncytia, during cell-cell fusion, irrespective of co-expression of p17, (Fig 7B), consistent with previous observations [32,50]. By contrast, cell-cell fusion was induced in DemKT1 cells only by the co-expression of FAST

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Regulation of viral replication by a viral factor in bat cells  (Figs 7B and S3), suggesting that the enhancement of fusion activity of FAST by p17 is specific to bat cell lines. To further confirm the role of p17 in the regulation of FAST fusion activity, we examined FAST-mediated cell-cell fusion activity in the presence of either wild-type or mutant p17. Expression of wild-type but not of either of the p17 mutants lacking nuclear localization activity induced cell-cell fusion in DemKT1 cells (Fig 7C). We also observed that cell-cell fusion was clearly induced by transient transfection of FLAG-tagged wild-type p17 but not its mutants in DemKT1 cells (S4A and S4B Fig). These results suggest that nuclear localization of p17 is required for induction of fusion activity of FAST in bat cells.

Enhancement of FAST function by p17 is required for viral replication in DemKT1 cells
To investigate the effects of the enhancement of FAST fusion activity by p17 on viral replication, we generated a recombinant virus that neither expressed FAST nor p17 (rsMB/FAST-p17-null) by disrupting the FAST start codon ( 27 ATG 29 !ATT) and creating a stop codon ( 39 TGC 41 !TGA) in an rsMB/p17-null background (S1 Table). A trans-complementation assay was then used to examine the effect of expression of p17 or FAST on viral replication. Vero and DemKT1 cells were transfected with expression plasmids encoding p17 and/or FAST prior to infection with rsMB/ FAST-p17-null at an MOI of 0.1 and expression of viral proteins and titers in cells were determined. In Vero cells, σC and σNS expression and viral titers were restored by the expression of FAST, while viral protein expression and infectious titer were little affected by the restoration of p17 expression (Fig 8A and 8B), indicating that FAST protein alone is required for efficient viral replication in Vero cells, consistent with previous observations [37]. By contrast, co-expression of both FAST and p17 was required for restoration of viral protein expression and viral titers in DemKT1 cells (Fig 8C and 8D). Together, these data indicate that the enhancement of FAST function by p17 is essential for viral replication in DemKT1 cells.

A p17 homologue from other bat-borne orthoreovirus enhances NBV replication in DemKT1 cells
Other fusogenic orthoreoviruses encode p17 homologues in gene segments encoding FAST proteins. ARV p17, for example, is encoded on the S1 gene segment, while the BRV and BroV

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Regulation of viral replication by a viral factor in bat cells p17 homologue, p16, is encoded on the S4 gene segment. To determine whether the p17 homologues have similar functions to NBV p17, we examined the activation of NBV FAST fusion activity in DemKT1 cells by co-expressing either NBV p17 or p17 homologues. ARV p17, BRV p16, and BroV p16 induced FAST-mediated cell-cell fusion and BroV p16, in

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Regulation of viral replication by a viral factor in bat cells particular, significantly increased cell-cell fusion (Fig 9A). We next performed complementation assays in DemKT1 cells infected with rsMB/p17-null using transient expression of the p17 homologues. We found that expression of BroV p16 but not of ARV p17 and BRV p16 restored viral replication in DemKT1 cells to a level comparable to that of wild-type NBV p17 (Fig 9B). Taken together, these data suggest that the regulation of NBV replication by the expression of p17 homologues in DemKT1 cells is virus species-specific.

Discussion
The S1 gene segment of the bat-borne fusogenic virus NBV is tricistronic, encoding three partially overlapping open reading frames: FAST (27-314 nt), p17 (277-705 nt), and σC (572-1567 nt) [38]. Our previous studies have shown that NBV nonstructural FAST and structural σC proteins play a critical role in viral replication and pathogenesis [37,45]. In the present study, we found that while p17 does not contribute to NBV replication in many human and animal cell lines or to pathogenicity in mice (Figs 1 and 2), replication of a p17-deficient virus

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Regulation of viral replication by a viral factor in bat cells is remarkably impaired in cells derived from the natural host of NBV (Fig 3). Bats are the natural host for many highly pathogenic viruses, including SARS coronavirus and Nipah virus, but these viruses cause no clinical signs of disease in bats in general [12][13][14][15], suggesting that virus and host have co-evolved a benign relationship. It has been reported that bat immunology is unique, with their immune system able to repress and allow viral replication [6,16,51,52]. While most reports have focused on the host factors responsible for the regulation of infection, the viral factors responsible for viral replication in bats remain largely unknown and, therefore, the functional analysis of NBV p17 as a potential host-dependent factor influencing replication in bat cells is important for understanding the mechanism responsible for symbiosis between viruses and natural hosts.
Using various p17 truncation and substitution mutant viruses, we have identified basic amino acids at positions 117, 118, 131, and 132 that are critical for the enhancement of NBV FAST and the regulation of viral replication (Figs 5 and 6). These C-terminal amino acids are essential for the nuclear localization of NBV p17. Two residues, K117 and R118, are conserved between NBV and ARV strains, while downstream K131 and R132 residues are conserved among NBV strains only. Recently, the basic K122 and R123 residues of ARV p17 (K117 and R118 in NBV) were reported to act as a nuclear localization signal for the induction of autophagy, correlated with an enhancement in viral replication [40,53]. Thus, it is possible that NBV p17 may contribute to the regulation of viral replication by a similar pathway in bat cells. The downstream KR motif, which is conserved among NBV strains may be required for NBV-specific regulation of replication in bat cells.
We found that NBV p17 promotes viral replication by enhancing cell-cell fusion activity of FAST in bat cells (Figs 7 and 8). However, how NBV p17 enhances the fusogenic activity of

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Regulation of viral replication by a viral factor in bat cells FAST in bat cells is still unknown. It is possible that p17 affects host transcription and controls the expression of host factors that associate with FAST, as nuclear localization of p17 in bat cells is essential for its function. While it is unknown whether NBV p17 interacts with nucleic acids or cellular transcription factors, an interaction between ARV p17 and cellular heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and lamin A/C has been reported and knockdown of these host factors inhibits the nucleocytoplasmic shuttling of ARV p17 [54]. We hypothesize that the interactions between NBV p17 and host factors such as hnRNP A1 and lamin A/C play a role in regulating nuclear localization of NBV p17 and directly or indirectly affect viral replication dependent on NBV p17 nuclear localization activity in bat cells.
A previous study reported that the cellular protein annexin A1 (AX1) interacts with reptilian orthoreovirus FAST-p14 protein and enhances cell-cell fusion in a calcium-dependent manner [55]. This interaction promotes membrane aggregation and is needed for fusion pore expansion mediated by FAST. AX1 similarly promotes syncytium formation by measles virus membrane fusion proteins F and H, suggesting that AX1 is necessary for syncytium formation. Thus, it is possible that NBV p17 may regulate expression and localization of host factors required for FAST-dependent cell-cell fusion such as AX1. Further studies are needed to understand which step of the cell-cell fusion process is regulated by p17 in bat cells. In the genus Aquareovirus, it is known that the Aquareovirus-C (AqRV-C) NS26 protein enhances the fusogenic activity of AqRV-C FAST-NS16 and co-localizes with lysosomes and lysosomeassociated membrane protein 1 (LAMP1) to stimulate syncytium formation by FAST-NS16 [56,57]. The vacuolar-targeting TLPK motif of NS26 is critical for this function. Although the motif is not conserved in NBV p17 protein, it is possible that NBV p17 promotes the modification of lysosomes to enhance FAST-mediated fusion activity and viral replication in bat cells. Further analyses of cellular protein expression, metabolites and organelle function are required to improve our understanding of NBV p17 function in bat cells.
On the other hand, there is a possibility that bat DemKT1 cells encode unique restriction factors that inhibit the induction of cell-cell fusion by FAST. Previous studies have shown that several cellular restriction factors can inhibit the replication of viruses; apolipoprotein B mRNAediting enzyme catalytic polypeptide-like 3G (APOBEC3G), for example, is efficiently incorporated into human immunodeficiency virus type 1 (HIV-1) particles, inhibiting viral replication by inducing G-to-A hypermutation in the viral cDNA during reverse transcription [58,59]. HIV-1 Vif protein can counteract this restriction by binding to APOBEC3G, leading to proteasomal degradation [58,60]. Tetherin, also known as BST-2, is a restriction factor that blocks the release of budded viral particles of retroviruses, such as HIV-1, and other enveloped viruses, including Ebola virus and vesicular stomatitis virus, by tethering virus particles to the plasma membrane [61][62][63]. Interestingly, the ability of these viral proteins to antagonize APOBEC3G and tetherin is species-specific [64][65][66][67]. Thus, it is possible that bat cells encode as-yet-unknown restriction factors associated with inhibition of FAST function that are disrupted by NBV p17.
NBV p17 enhances FAST function, regulating viral replication in a virus species-specific (Fig 9). Interestingly, a p17 homologue in another bat-borne orthoreovirus, BroV, efficiently enhances NBV FAST function in bat cells and is capable of restoring replication in p17-deficient NBV (Fig 9). ARV p17, which shares greater homology to NBV p17 than BroV p16, was incapable of restoring replication in p17-deficient viruses, suggesting potential co-evolution between fusogenic reoviruses and host species. While amino acid sequences differ, it is possible that the structures of NBV p17 and BroV p16 protein share similar characteristics. Further comparative studies regarding the structure and function of both proteins will promote understanding of fusogenic reovirus-host interactions. Bat-borne fusogenic reovirus p17 proteins have been suggested to control expression and/or localization of positive or negative host factors associated with FAST-dependent cell-cell fusion activity for efficient replication in bat cells. However, overexpression of NBV p17 promotes excessive FAST-mediated cell-cell fusion and viral replication, resulting in host cell damage. This would adversely affect both NBV and bats, suggesting that an as of yet unidentified mechanism between NBV and bats regulates the expression level of p17; for instance, the translation machinery of p17 could be the target of such a mechanism. NBV p17 is encoded by the tricistronic S1 gene segment and p17 is encoded as a second ORF between FAST and σC ORFs. A previous study demonstrated that leaky scanning is the predominant mechanism for ARV p17 translation from tricistronic S1 mRNA [68]. Such leaky scanning may affect the regulation of NBV p17 expression and play an important role in viral replication in bat cells. It is possible that other mechanisms could allow alternative bat-specific translation initiation of p17 by interacting or controlling host translational factors. The identification of bat factors interacting with NBV FAST and NBV p17, and understanding the translation mechanism of the three genes encoded by S1 gene segments, will be important for further analysis of the NBV replication regulation system in bats.
The present study shows that NBV p17 regulates viral replication in a host-specific manner. Further analysis of fusogenic reovirus p17 and FAST proteins in bat-borne viruses will contribute greatly to our understanding of the relationship between reservoir, virus, and the regulation of viral replication.

Electrophoretic analysis of dsRNA genomes
Viral genomic RNA was extracted from virions using Sepasol RNA I Super G (Nacalai Tesque) and mixed with an equal volume of 2 × sample buffer (125 mM Tris-HCl pH 6.8, 10% sucrose). The genomic RNA was separated using a 10% precast polyacrylamide gel (Atto) and visualized by ethidium bromide staining.

Cell death assay
DemKT1 cells (3 × 10 4 ) were plated in 96-well plates (Corning) and incubated overnight. The cells were infected with viruses at an MOI of 0.1 PFU/cell and incubated at 37˚C for 1 h. Subsequently, the supernatant was removed, and the cells were washed with PBS and then incubated

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Regulation of viral replication by a viral factor in bat cells in RPMI (100 μL; 2% FBS) for various intervals. WST-1 (Roche) (20 μL) diluted to 10% in PBS was added to the cells and the cells were incubated at 37˚C for 1 h. Cell viability was calculated by quantifying the cleavage of WST-1 to formazan by cellular succinate-tetrazolium reductase. Formazan was quantitated by measuring absorbance at A 450 nm-A 690 nm using a Power-ScanHT (DS Pharma).

Immunofluorescence analysis
p17 expression plasmids were transfected into DemKT1 cells (1×10 5 ) on glass coverslips. At 25 h post-transfection, samples were fixed with 4% paraformaldehyde and permeabilized using 0.5% Triton-X. DemKT1 cells (2×10 5 ) and Vero cells (1×10 5 ) were infected with virus at an MOI of 0.1 PFU/cell at 37˚C for 1 h, and cells were washed and incubated in growth media. At 13-26 h post-infection, samples were fixed with 4% paraformaldehyde and permeabilized using 0.5% Triton-X. Fixed cells were incubated with antibodies and DAPI or Hoechst, and the samples were mounted using ProLong diamond antifade mountant (Thermo Fisher scientific). Images were acquired with a FluoView FV1000 laser scanning confocal microscope (Olympus).

Subcellular fractionation
pCXN2-NBV p17-FLAG or p17 mutant plasmids were transfected into DemKT1 cells (3×10 5 ). At 24 h post-transfection, cells were washed with PBS and collected by trypsin digestion. The cells were resuspended in suspension buffer (10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , and 2.5% mercaptoethanol) and collected by centrifugation (6,000×g, 5 min, 4˚C). The cells were subsequently lysed in 200 μL of suspension buffer supplemented with 1.25% NP-40 (Nacalai Tesque) on ice, vortexing every minute for 5 min. Samples were centrifugated at 6,000×g for 15 min at 4˚C and the supernatant was collected as the cytoplasmic fraction. The pellet was washed with suspension buffer 3 times and lysed in 200 μL of RIPA lysis buffer. The lysed pellet was centrifugated at 6,000×g for 5 min at 4˚C and the supernatant was collected as the nuclear fraction.

Cell-cell fusion assay
Cells were plated at an appropriate concentration (DemKT1 cells at 4 × 10 5 cells/well and Vero cells at 2 × 10 5 cells/well in 12-well plates; YubFKT1 cells at 1 × 10 5 cells/well in 24-well plates) and transfected with p17 and/or FAST expression vectors. At 16-26 h post-transfection, cells were fixed with methanol and stained with Giemsa's Stain Solution (Nacalai Tesque), and the number of fusion cells were counted in random microscopic fields (100×total magnification).

Complementation of viral replication in cells infected with a p17 mutant virus
To examine viral protein expression, pCXN2-NBV p17 (1 μg) was transfected into DemKT1 cells (2.5 × 10 5 ) and, at 10 h post-transfection, infected with rsMB/p17-null at an MOI of 1 PFU/cell. After 1 h, the cells were washed with PBS and incubated in growth media for 16 h at 37˚C. The cells were lysed and viral proteins were detected using antibodies against NBV proteins. To examine viral replication, pCXN2-NBV p17 (0.5 μg) was transfected into DemKT1 cells (5 × 10 4 ) and, at 24 h post-transfection, infected with rsMB/p17-null at an MOI of 0.1 PFU/cell. After 1 h, the cells were washed with PBS and incubated in growth media for 24 h at 37˚C. The cells were collected and viral titers were determined by plaque assay on a monolayer of L929 cells.

Complementation of viral replication in cells infected with a FAST-p17 mutant virus
To examine viral protein expression, pCXN2-NBV p17 (1 μg) was transfected into DemKT1 cells (2.5 × 10 5 ) or Vero cells (1 × 10 5 ) with or without pCAG-NBV FAST (1 μg). At 3 h posttransfection, the DemKT1 cells were infected with rsMB/FAST-p17-null at an MOI of 0.1 PFU/cell. After 4 h of incubation, the cells were washed with PBS and incubated for 17 h at 37˚C. The Vero cells were infected with the virus at an MOI of 0.1 PFU/cell at the same time of transfection, incubated for 12 h at 37˚C, and lysed. Viral proteins were detected using antibodies against NBV proteins. To examine viral replication, pCXN2-NBV p17 (0.5-1.0 μg) was transfected into DemKT1 cells (1 × 10 5 ) or Vero cells (5 × 10 4 ) with or without pCAG-NBV FAST (0.5-1.0 μg). At 3 h post-transfection, the cells were infected with rsMB/FAST-p17-null at an MOI of 0.1 PFU/cell. After 1 h of incubation, the cells were washed with PBS and incubated for 17-20 h at 37˚C. The supernatant was collected and viral titers were determined by plaque assay on a monolayer of L929 cells.

Statistical analysis
Each data point is expressed as the mean of at least three samples and error bars indicate the standard deviation. Statistical significance was determined via Welch's t-test, log rank test, or one-way ANOVA followed by Dunnett's test where indicated. p values < 0.01 or 0.05 were considered statistically significant.